e-PG Pathshala for Biophysics, MHRD project, UGC PI: Prof M.R. Rajeswari, A.I.I.M.S., New Delhi Paper 05: Molecular Enzymology and Protein Engineering Module No. 13: Mechanism and Kinetics of Competitive inhibition Content writer: Dr. Vishvanath Tiwari Department of Biochemistry, Central University of Rajasthan, Ajmer-305817 Objective: Objective of the present module is to understand the mechanism and kinetics of the competitive inhibition as well as role of dissociation constant of inhibitor in drug designing. We will also discuss the different examples of the competitive inhibition. This module is divided into following sections- 1. Introduction 2. Competitive inhibition 2.1 Mechanism of competitive inhibitor 2.2 Kinetics of competitive inhibitor 2.3 Determination of dissociation constant for competitive inhibitor 2.4 Examples of competitive inhibitors 4. Significance of the competitive inhibition in drug designing 3. Summary 4. Question 5. Resources and suggested reading 1. Introduction: The negative regulator or enzyme inhibitor can reduce the rate of enzyme-catalyzed reaction. Inhibition of the enzyme could be significant in term of inhibiting the crucial Content by Dr. Vishvanath Tiwari 1 enzymatic pathways. Enzyme inhibitions are irreversible, suicide, feedback and reversible inhibition. Reversible inhibition involves weak non-covalent interactions between enzyme and inhibitors. The non-covalent interaction involves hydrogen bonding, hydrophobic interactions, van der Waal’s forces and salt bridges. The cumulative effects of these interactions result into strong interactions. Because of weak non-covalent interactions, reversible inhibitor can be separated from the enzymes therefore the name reversible inhibition is given. On the basis of effect of varying concentration of enzyme’s substrate on the inhibitor, Dr. W W Cleland classified reversible inhibition into four groups i.e competitive, non-competitive, un- competitive and mixed inhibition. In the present module we will discuss about the characteristics of the competitive inhibition, kinetics of inhibition and different example of the competitive inhibition. 2. Competitive inhibition: 2.1 Mechanism of competitive inhibitor Competitive inhibitors have structural similarity with the substrate therefore compete with the substrate for binding on the active site of the enzyme. It is well known that enzyme have only one active site therefore two molecules cannot bind at the same site therefore there is competition between competitive inhibitor and substrate. The binding affinity of the competitive inhibitor and the substrate is the deciding factor for the selection of candidate for the binding on active site. Therefore, at a given time, free enzyme either can form ES (Enzyme-substrate) complex or EI (enzyme- inhibitor) complex but Enzyme-substrate-inhibitor (ESI) complex cannot be formed (Figure 1). Hence, there is a competition between substrate and competitive inhibitors for binding on the enzyme i.e. both are mutually exclusive, therefore the name competitive inhibition is given to this type of inhibition. The competitive inhibitor may be non-metabolizable analog or derivate of the true enzyme o r alternate substrate of the enzyme or a product of the reaction. Increasing substrate concentration can reverse the competitive inhibition. Content by Dr. Vishvanath Tiwari 2 Figure 1: Model of the competitive inhibition 2.2 Kinetics of competitive inhibitor The competitive inhibitor will bind with the free enzyme and inhibit the binding of the substrate to the active site. During the mutual exclusion, if the substrate binds before the inhibitor, then it will be converted into the product, while if competitive inhibitor will bind to the enzyme before the substrate, then substrate will not bind to the enzyme and no product formation takes place. Based on this information, it can be stated that the competitive inhibitor will alter the binding of substrate to the enzyme i.e alter Km of the enzyme; while product formation of ES complex will not be altered i.e Vmax will not be altered. At any concentration of the competitive inhibitor, portions of enzyme exist in EI forms that have no affinity for the substrate (highest Km) and hence there is an increase in the Km of the enzyme. Increase in the Km does not mean that EI complex have lower affinity for the substrate but they have no affinity at all for the substrate, therefore apparent increase in the Km in the presence of the competitive inhibitor results from the full affinity (ES) and no affinity (EI). In the presence of competitive inhibitor, Vmax remains unchanged as ES complex formed is converted into the product, but in the presence of the competitive inhibitor a much greater concentration of the substrate concentration is required to attain any fraction of Vmax. Content by Dr. Vishvanath Tiwari 3 The Michaelis–Menten kinetic equation and plot can be derived for the competitive inhibition and written as Where vo is the initial velocity, Vmax is the maximum velocity, Km is Michaelis-Menten constant, [I] is the inhibitor concentration, [S] substrate concentration and Ki is the dissociation constant of the inhibitor with the enzyme. The modified Km is known as Km apparent and is equal to the is considered as an [I] dependent statistical factor describing the distribution of enzyme between E and EI forms. It is also represented by (alpha) which is equal to . Figure 2: Comparative Michaelis–Menten kinetic plot for the competitive inhibition. Content by Dr. Vishvanath Tiwari 4 Similarly, the Lineweaver–Burk equation for the competitive inhibition can be derived from the Michaelis-Menten and written as Based on the Lineweaver-Burk plot following plot can be drawn Figure 3: Comparative Lineweaver–Burk plot for the competitive inhibition. 2.3 Determination of the dissociation constant of competitive inhibitor Ki is the dissociation constant of the inhibitor. It can be calculated either from the slope of the plot or by the 1/[S] axis intercept. Km apparent can be calculated by the reciprocal of the x-axis intercept in the presence of the inhibitor. Ki of an inhibitor can be calculated using the equation Km apparent is the Km in the presence of competitive inhibitor, Km is the km in absence of the inhibitor, [I] is the inhibitor concentration. Therefore, if we can calculate the apparent Km in the presence of the competitive inhibitor, Km in the absence of inhibitor and if we know the concentration of the inhibitor, then we can calculate the dissociation constant of the competitive inhibitor. Ki of the inhibitor is Content by Dr. Vishvanath Tiwari 5 inversely proportional to the binding affinity of the competitive inhibitor and Enzyme. As the inhibitor concentration increases, the slope of the plot will increase and the intercept on the 1/[S] axis move closer to the zero i. e. the apparent Km continuously increases. 2.4. Examples of the competitive inhibitors Competitive inhibition is most common reversible inhibition. There are many examples of competitive inhibition, which will be discussed. Example 1: Alcohol dehydrogenase converts ethanol into acetaldehyde. This reaction also converts NAD into NADH that is utilized by the body for the production of the ATPs. Methanol is the competitive inhibitor of the alcohol dehydrogenase because of the structural homology of methanol with the ethanol. Methanol is converted into the formaldehyde that is responsible for the lethal effect of methanol poisoning. Example 2: Similarly, Succinate dehydrogenase is an important enzyme of the TCA cycle; hence malonate is used as an inhibitor of the TCA cycle. This enzyme catalyzes the conversation of succinate into fumarate by using FAD as co-enzyme. Malonate (propanedioate) has structural similarity with the succinate but lack CH2- CH2 group that is required for the activity of succinate dehydrogenase that uses FAD as co-enzyme; therefore malonate is a competitive inhibitor of succinate dehydrogenase because it can bind to the succinate dehydrogenase due to its structural similarity but unable to convert malonate into the its product, hence malonate is a competitive inhibitor of succinate dehydrogenase. Content by Dr. Vishvanath Tiwari 6 Example 3: During the TMP synthesis from UMP, the methylation agents i.e. tetrahydrofolate is used, which is converted into dihydrofolate during the reaction catalyzed by thymidylate synthase. Dihydrofolate must be again converted back into the tetrahydrofolate by the enzyme dihydrofolate reductase to complete the reaction cycle. Methotrexate and aminopterin are the structural homolog of the folic acid; therefore it can be used as competitive inhibitor of the dihydrofolate reductase. Hence in the presence of this inhibitor, TMP biosynthesis is inhibited due to absence of tetrahydrofolate. This is the reason why methotrexate is used as anti-cancer drug and aminopterin is inhibitor of de-novo synthesis of DNA. Aminopterin is important component of the HAT media that are used to produce monoclonal antibody. Example 4: Cholesterol synthesis takes place from the acetic acid via intermediate such as HMGCoA, mevalonate, squalene and lanosterol. Cholesterol synthesis require important enzyme called HMG-COA reductase that converts HMGCoA (3- hydroxy-3-methyl-glutaryl-CoA) into mevalonate by reduction that uses NADPH. Mevalonate is precursor for the biosynthesis of the cholesterol hence the synthesis of the mevalonate is very important for the synthesis of cholesterol. Statin group of drugs are structure homolog of the HMG-CoA,